Proteins form stable and dynamic multi-subunit complexes under different physiological conditions to maintain cell viability and normal cell homeostasis. Detailed knowledge of protein interactions and protein complex structures is fundamental to understanding how individual proteins function within a complex and how the complex functions as a whole. However, structural elucidation of large multi-subunit protein complexes has been difficult due to lack of technologies which can effectively handle their dynamic and heterogeneous nature. Traditional biophysical methods such as nuclear magnetic resonance (NMR) analysis and X-ray crystallography can yield detailed information on protein structures; however, NMR spectroscopy requires large quantities of pure protein in a specific solvent while X-ray crystallography is often limited by the crystallization process.
Current biochemical methods are not very efficient to analyze system-level or large-scale protein interaction networks. Most of the studies utilize a technique called “co-immunoprecipitation,” where a protein is isolated along with its interacting partners (protein complexes) by using an antibody or by incorporating an affinity group in the protein which can be used as a hook to selectively purify that protein. This method is applicable for very strong and stable interactions, but most of the cellular interactions are very transient and weak, and during the purification process these interactions get lost completely. Besides, this identification is very qualitative and does not put emphasis on the protein-to-protein interaction domain.
One chemistry-based fixation method combined with mass spectrometry technology utilizes a crosslinker to stabilize proteins with its interaction partners (protein complexes) by using certain side chains of proteins)) before performing cell lysis. Crosslinkers can fix the nearby proteins or protein complexes by chemical reactions and hold them tightly so they will not detach after cell lysis and will not be affected by the subsequent strict purification conditions. In addition, a crosslinker reacts within a limited distance; hence, protein reactive sites can be measured by calculating the distances of the reactive sites. This method can identify large-scale protein interactions and it can identify protein structures in their native biological conditions.
Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multi-subunit protein complexes, this method has proven challenging due to technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. The universal use of this technology is hindered due to several bottlenecks. Previously disclosed crosslinking strategies generate an enormous amount of mass spectrometry data which is extremely difficult to analyze by routine software tools. Finding these interactions in large datasets is akin to finding a needle in a haystack. Examples of known crosslinking strategies include selective enrichment using click chemistry with alkyne-tagged (Chowdhury, et al., Anal Chem., 81:5524-5532 (2009)); affinity enrichment combined with isotopic coding and CID cleavage (Petrotchenko, et al., MCP, 10:M110 001420 (2011); MS-cleavable reagents (Soderblom, et al., Anal Chem. 78:8059-8068 (2006)); and Tang, et al., Anal Chem., 77:311-318 2005)); crosslinking using the amine-reactive disuccinimidyl suberate (DSS) (Greber, et al., Nature, 515:283-286 (2014)) and Greber, et al., Science, 348:303 (2015)); lysine-targeted enrichable cross-linker containing a biotin tag (Tan, et al., eLife, 5 (2016)); in vivo cross-linking (X) assisted bimolecular tandem affinity purification strategy (Yu, Molecular & Cellular Proteomics, 15(7):2279-92 (2016)); and amidinating protein cross-linker, DEST (diethyl suberthioimidate) (Lauber, et al., Molecular & Cellular Proteomics, 11(12): 1965-76 (2012)) and acidic residue reactive cleavable cross-linker (Anal Chem. 2016 Aug. 16; 88(16): 8315-8322).
There remains a need to make crosslinking technology very amenable for analyzing large-scale protein interactions, through the design of more effective chemical crosslinkers with innovative features, which will help reduce the complexity of mass-spectrometry data from large-scale protein interactions, and easy to analyze software tools.
It is an object of the present invention to provide chemical crosslinkers which reduce the complexity of mass-spectrometry data.
It is still an object of the present invention to provide a method of making chemical crosslinkers which reduce the complexity of mass-spectrometry data.
It is also an object of the present invention to provide a method for identifying crosslinked peptides with improved fidelity.